Methods for selectively removing counterions from compounds and compounds derived from such methods

ABSTRACT

Methods of selectively removing counterions from compounds are disclosed. Compounds derived from the methods, such as the compound S-[2-[( 1 -Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterion with between zero and up to 2 molar equivalents of hydrochloride are also disclosed.

[0001] Priority is claimed from U.S. Provisional Application Ser. No.60/453,798, filed Mar. 11, 2003 incorporated herein by reference

FIELD OF THE INVENTION

[0002] The present invention provides novel methods for removingcounterions from compounds, especially useful for preparing ionic saltsfrom zwitterionic compounds. The present invention also comprises novelcompounds useful in the treatment of disease, and more particularly anovel intermediate for preparing salts ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, and pharmaceuticalcompositions thereof, for the treatment of conditions involving aninappropriate expression of nitric oxide from the inducible isoform ofnitric oxide synthase.S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine is described andclaimed in commonly assigned U.S. Pat. No. 6,403,830, hereinincorporated by reference.

BACKGROUND OF THE INVENTION

[0003] Dipolar compounds, that is to say, molecules that exhibit bothone or more positive and negative formal charges within the samestructure, are known as zwitterions. At a pH specific to a particularzwitterion, known as the isoelectric point (pI), the net charge of thatzwitterion is zero. In aqueous solution, zwitterions are typically leastsoluble at the isoelectric point. In biological systems, α-amino acidsare among the most common zwitterionic molecules. The simplest aminoacid is glycine, which has no side groups. In acidic solutions, both theα-amino group and the carboxylic acid group of glycine are protonated.At the isoelectric point, the α-amino group is protonated, but thecarboxyl group is ionized. In basic solutions, both the α-amino groupand the carboxyl group are deprotonated. Some natural amino acids, suchas lysine, arginine and histidine, have basic side chains, and thusundergo multiple ionization states. Most naturally occurring amino acidsmay be precipitated by titration with appropriate acidic or basicsolutions, such as hydrochloric acid or aqueous sodium hydroxide,respectively, until the isoelectric point, and hence the lowestsolubility are achieved. In addition, removal of excess cationiccounterion from such a titration may be performed by adding a sufficientamount of strong base. For example, to remove hydrochloride cations froman amino acid, an amount of sodium hydroxide may be added to form thesodium salt of the amino acid.

[0004] Nitric oxide (NO) is a bioactive free radical gas produced by anyone of several isoforms of the enzyme nitric oxide synthase (NOS). Thephysiological activity of what was later identified as NO was initiallydiscovered in the early 1980's when it was found that vascularrelaxation caused by acetylcholine is dependent on the presence of thevascular endothelium. The factor derived from the endothelium, thencalled endothelium-derived relaxing factor (EDRF), that mediates suchvascular relaxation is now known to be NO that is generated in thevascular endothelium by one isoform of NOS. The activity of NO as avasodilator has been known for well over 100 years. In addition, NO isthe active species derived from known nitrovasodilators includingamylnitrite, and glyceryltrinitrate. Nitric oxide is also an endogenousstimulator of soluble guanylate cyclase (cGMP), and thus stimulates cGMPproduction. When NOS is inhibited by N-monomethylarginine (L-NMMA), cGMPformation is completely prevented. In addition to endothelium-dependentrelaxation, NO is known to be involved in a number of biological actionsincluding cytotoxicity of phagocytic cells and cell-to-cellcommunication in the central nervous system.

[0005] The identification of EDRF as NO coincided with the discovery ofa biochemical pathway by which NO is synthesized from the amino acidL-arginine by the enzyme NO synthase. There are at least three types ofNO synthase as follows:

[0006] (i) a constitutive, Ca++/calmodulin dependent enzyme, located inthe brain, that releases NO in response to receptor or physicalstimulation;

[0007] (ii) a Ca++ independent enzyme, a 130 kD protein, which isinduced after activation of vascular smooth muscle, macrophages,endothelial cells, and a number of other cells by endotoxin andcytokines; and

[0008] (iii) a constitutive, Ca++/calmodulin dependent enzyme, locatedin the endothelium, that releases NO in response to receptor or physicalstimulation.

[0009] Once expressed, inducible nitric oxide synthase (hereinafter“iNOS”) generates NO continuously for long periods. Clinical studieshave shown that NO production and iNOS expression are increased in avariety of chronic inflammatory diseases, such as rheumatoid andosteoarthritis (see, e.g, Mclnnes I. B. et al., J. Exp. Med. 184:1519(1996)), inflammatory bowel disease (see, e.g, Lundberg J. O. N. et al.,Lancet 344:1673, (1994)), and asthma (see, e.g., Hainid, Q. et al.,Lancet 342:1510 (1993)), and iNOS is implicated as a major pathologicalfactor in these chronic inflammatory diseases.

[0010] Thus, inhibition of excessive NO production by INOS is likely tobe anti-inflammatory. However, since the production of NO from eNOS andnNOS is involved in normal physiology, it would be desirable for any NOSinhibitor that is used for treating inflammation be selective for iNOS,so that normal physiological modulation of blood pressure byeNOS-generated NO, and non-adrenergic, non-cholinergic neuronaltransmission by nNOS-generated NO would remain unaffected.

[0011] With all pharmaceutical compounds and compositions, the chemicaland physical stability of a drug compound is important in the commercialdevelopment of that drug substance. Such stability includes thestability at ambient conditions, especially to moisture and understorage conditions. Elevated stability at different conditions ofstorage is needed to predict the different possible storage conditionsduring the lifetime of a commercial product. A stable drug avoids theuse of special storage conditions as well as frequent inventoryreplacement. A drug compound must also be stable during themanufacturing process which often requires milling of the drug toachieve drug material with uniform particle size and surface area.Unstable materials often undergo polymorphic changes. Therefore, anymodification of a drug substance which enhances its stability profileprovides a meaningful benefit over less stable substances.

[0012] Several inhibitors of iNOS have been described, such as, forexample, S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine, which isdescribed and claimed in commonly assigned U.S. Pat. No. 6,403,830. Thatcompound, however, is an amorphous solid. It would be desirable,therefore, to provide a crystalline solid form of an iNOS inhibitor suchas S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine.

[0013] Other iNOS inhibitors compounds that are synthetic amino acidanalogs that include amidine functional groups include:S-[2-[(1-Iiminoethyl)amino]ethyl]-2-methyl-L-cysteine, dihydrochloride;S-[2-(ethanimidoylamino)-1-methylethyl]cysteine;(2S,5E)-2-amino-6-fluoro-7-[(1-iminoethyl)amino]-5-heptenoic acid,dihydrochloride; (S,E)-2-amino-2-methyl-6-[(1-iminoethyl)amino]-4-hexenoic acid,dihydrochloride;(2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic acid,dihydrochloride; and(2S,5E)-2-amino-2-methyl-6-fluoro-7-[(1-iminoethyl)amino]-5-heptenoicacid, dihydrochloride.

[0014] Unfortunately, traditional methods of removal of cationiccounterions by titration with a strong base generally results indecomposition of the amidine functional group of these compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a schematic ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine titration curve,showing all ionization states;

[0016]FIG. 2 is a graphical representation of titration curves ofS-[2-[(1-Iminoethyl)amino]etbyl]-2-methyl-L-cysteine in water withIRA-400(OH) anion exchange resin. Diamond is pH and square (dashed line)is S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine (% initial, byion chromatography);

[0017]FIG. 3 is a graphical representation of titration curves ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine in water withIRA-400(OH) anion exchange resin. Diamond is pH and triangle (brokenline) is chloride (by ion chromatography);

[0018]FIG. 4 Shows titration curves ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine in water withIRA-400 anion exchange resin; and

[0019]FIG. 5 shows the relevant binding data associated with increasingpH.

SUMMARY OF THE INVENTION

[0020] In a broad sense, present invention is directed to a method forthe removal of salt counterions from a compound using an appropriate ionexchange medium. In one embodiment, the ion exchange medium is a resin.In another embodiment, the ion exchange medium is a membrane. In anotherembodiment, the counterion is a cation, and the ion exchange resin is ananionic resin. In another embodiment, the counterion is an anion, andthe ion exchange resin is a cationic resin. In another embodiment, thecompound from which the counterion is to be removed is a zwitterion. Inanother embodiment, the method is performed with ion exchange resin in asingle stirred vessel. In another embodiment, method is performed withion exchange resin in several batches in a plurality of stirred vesselsin series, with intermediate filtering of the resin and replacing withfresh resin. In another embodiment, the zwitterionic compound from whichthe counterion is to be removed is a synthetic amino acid analog. Inanother embodiment of the invention, the synthetic amino acid includesan amidine functional group. In another embodiment of the presentinvention, there is provided method of makingS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterioncharacterized by having stoichiometrically less than 0.5 equivalents ofhydrochloride ion toS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, comprisingobtaining a source ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine dihydrochloride,and removing sufficient hydrochloric acid to obtain theS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterion havingstoichiometrically less than 0.5 equivalents of hydrochloride ion toS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine.

[0021] Several selective inhibitors of the inducible isoform of nitricoxide synthase have been discovered. Among these iNOS inhibitors arethose compounds that are synthetic amino acid analogs that includeamidine functional groups, such as, for example:S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, dihydrochloride;S-[2-(ethanimidoylamino)-1-methylethyl]cysteine;(2S,5E)-2-amino-6-fluoro-7-[(1-iminoethyl)amino]-5-heptenoic acid,dihydrochloride; (S,E)-2-amino-2-methyl-6-[(1-iminoethyl)amino]-4-hexenoic acid,dihydrochloride;(2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic acid,dihydrochloride; and(2S,5E)-2-amino-2-methyl-6-fluoro-7-[(1-iminoethyl)amino]-5-heptenoicacid, dihydrochloride. The methods of the present invention are usefulin partially or completely removing salt counterions from thesecompounds, especially for the replacement of counterions withalternative counterions, and mixtures of counterions, for thecrystallization of these compounds. The methods of the present inventionare particularly suited to compounds of this type, because traditionalmethods of removal of cationic counterions by titration with a strongbase generally results in decomposition of the amidine functional groupof these compounds. The methods of the present invention are also usefulin partially removing counterions from compounds. Partial removal ofcounterions results in stoichiometrically different proportions ofcounterion to compound. Thus, one embodiment of the present inventionprovides a method to treat a salt compound with an appropriate ionexchange medium to remove a sufficient amount of a salt counterion suchthat stoichiometrically less than 0.5 equivalents of the counterion tothe compound remains. Another embodiment of the present inventionprovides a method of makingS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterioncharacterized by stoichiometrically less than 0.5 equivalents ofhydrochloride ion toS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine comprisingobtaining a source ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine dihydrochloride,and removing sufficient hydrochloric acid to obtain theS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterion havingstoichiometrically less than 0.5 equivalents of hydrochloride ion toS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine.

[0022] The present invention is also directed to a novel intermediate,S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, useful in thepreparation of salts ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, a process forpreparing the novel intermediate compound, and methods of using saidnovel intermediate compound. The present intermediate compound possessesuseful nitric oxide synthase inhibiting activity, and is useful in thetreatment or prophylaxis of a disease or condition in which thesynthesis or oversynthesis of nitric oxide forms a contributory part.

[0023] The present novel intermediate itself can be used to treatdiseases involving cartilage degeneration, which takes place in certainconditions such as arthritis. Accordingly, conditions in which there isan advantage in inhibiting NO production from L-arginine includearthritic conditions such as rheumatoid arthritis, osteoarthritis, goutyarthritis, juvenile arthritis, septic arthritis, spondyloarthritis,acute rheumatic arthritis, enteropathic arthritis, neuropathicarthritis, and pyogenic arthritis. In addition, NO-induced depression ofchondrocyte respiration could modulate matrix loss and secondarycartilage mineralization in arthritis, in particular osteoarthritis.

[0024] Other conditions for which the present intermediate may be usefulinclude chronic or inflammatory bowel disease, cardiovascular ischemia,diabetes, congestive heart failure, myocarditis, atherosclerosis,migraine, glaucoma, aortic aneurysm, reflux esophagitis, diarrhea,irritable bowel syndrome, cystic fibrosis, emphysema, asthma,bronchiectasis, hyperalgesia, cerebral ischemia, thrombotic stroke,global ischemia (secondary to cardiac arrest), multiple sclerosis andother central nervous system disorders mediated by NO, for exampleParkinson's disease and Alzheimer's disease. Further neurodegenerativedisorders in which NO inhibition may be useful include nervedegeneration and/or nerve necrosis in disorders such as hypoxia,hypoglycemia, epilepsy, and in external wounds (such as spinal cord andhead injury), hyperbaric oxygen convulsions and toxicity, dementia e.g.pre-senile dementia, and AIDS-related dementia, Sydenham's chorea,Huntington's disease, Amyotrophic Lateral Sclerosis, Korsakoffs disease,imbecility relating to a cerebral vessel disorder, sleeping disorders,schizophrenia, depression, depression or other symptoms associated withPremenstrual Syndrome (PMS), anxiety and septic shock.

[0025] The present intermediate may also be used where nitric oxideinhibition may also play a role in the treatment, such as pain includingsomatogenic (either nociceptive or neuropathic), both acute and chronic.The present compounds could be used in any situation that a common NSAIDor opioid analgesic would traditionally be administered.

[0026] Still, other disorders that may be treated by inhibiting NOproduction with the present compounds include opiate tolerance inpatients needing protracted opiate analgesics, and benzodiazepinetolerance in patients taking benzodiazepines, and other addictivebehavior, for example, nicotine and eating disorders. The presentcompound may also be useful as antibacterial agents.

[0027] Further conditions in which the present intermediate may be usedto inhibit NO production from L-arginine include systemic hypotensionassociated with septic and/or toxic shock induced by a wide variety ofagents; therapy with cytokines such as TNF, IL-1 and IL-2; and as anadjuvant to short term immunosuppression in transplant therapy.

[0028] The present novel intermediate may also be useful in thetreatment of ocular conditions (such as ocular hypertension retinitisuveitis), systemic lupus erythematosis (SLE), glomerulonephritis,restenosis, inflammatory sequelae of viral infections, acute respiratorydistress syndrome (ARDS), oxidant-induced lung injury, IL2 therapy suchas in a cancer patient, cachexia, immunosuppression such as intransplant therapy, disorders of gastrointestinal motility, sunburn,eczema, psoriasis, gingivitis, pancreatitis, damage to thegastrointestinal tract resulting from infections, cystic fibrosis,treatment to a dysfunctional immune system such as an adjuvant to shortterm immunosuppression in organ transplant therapy, induction of labor,adenomatous polyposis, controlling tumor growth, chemotherapy,chemoprevention and bronchitis.

[0029] The present invention is also directed to pharmaceuticalcompositions for the treatment of pain, asthma and other airwaydisorders, cancer, arthritis, ocular disorders including retinopathiesand glaucoma, inflammation related disorders including irritable bowelsyndrome, and other disorders in which an excessive production of nitricoxide plays a role, which comprises a therapeutically effective amountof S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine together with apharmaceutically acceptable carrier, diluent or vehicle.

[0030] Besides being useful for human treatment, this compound is alsouseful for veterinary treatment of companion animals, exotic animals andfarm animals, including mammals, rodents, and the like. More preferredanimals include horses, dogs, and cats.

DETAILED DESCRIPTION

[0031] Definitions

[0032] The term “counterion,” as used herein, means an ion that ischemically associated with a compound, usually, but not necessarily, byionic bonding.

[0033] The term “DBU” means the bicyclic amidine 1,8-diazabicyclo[5.4.0]undecene.

[0034] The term “DMSO” means dimethyl sulfoxide.

[0035] The term “fresh resin” as applied to an ion exchange resin, andcan mean both previously unused ion exchange resin, as well as ionexchange resin that has been used and subsequently regenerated.

[0036] The term “HPLC” means high pressure liquid chromatography.

[0037] The term “ion exchange medium,” as used herein, means afunctionalized, insoluble support capable of selectively removing acounterion from a compound. Non-limiting examples of ion exchange mediainclude ion exchange resins and ion exchange membranes.

[0038] The term “NMR” means nuclear magnetic resonance, and may apply tonuclear magnetic resonance spectroscopy.

[0039] The term “purity” herein, unless otherwise qualified, means thechemical purity of a compound, such as, for example,S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, according toconventional HPLC assay.

[0040] The term “synthetic amino acid” means an amino acid that does notnormally occur in nature.

[0041] The term “synthetic amino acid analog” means a synthetic aminoacid that has been further modified or rationally designed to acquire adesired property.

ILLUSTRATIVE EXAMPLES

[0042] The following illustrative examples are not intended to belimiting in scope, and should not be interpreted to limit the scope ofthe appended claims. While the compound illustrated in the followingexamples is S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine, themethods employed are applicable to a wide variety of compounds, areparticularly useful in the partial or complete removal of counterionsfrom linear amidine nitric oxide synthase inhibiting compounds.

[0043] A method to makeS-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine dihydrochloride isdescribed in commonly assigned U.S. Pat. No. 6,403,830, incorporatedherein by reference.

[0044] Briefly, synthesis ofS-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine dihydrochloride maybe performed as in the following Example 1:

Example 1

[0045]

[0046] S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine,dihydrochloride

Example-1A N-Boc-cysteamine

[0047]

[0048] A 3L 4-neck RB flask was purged with nitrogen for 20 min and thencharged sequentially with 2-aminoethanethiol hydrochloride (113.6 g, 1mol), di-tert-butyl-dicarbonate (218.3 g, 1 mol) and 500 mL of toluene.The mixture was cooled with an ice-water bath and purged with nitrogenfor 10 min. Sodium hydroxide (2.5N, 880 mL, 2.2 mol) was added to thestirring mixture in about 1.5 h at between 0 and 11° C. After theaddition of sodium hydroxide was complete, the cooling bath was removedand the resulting reaction mixture was allowed to warm up to roomtemperature and stirred at ambient temperature overnight. This provideda solution of the title product.

Example-1B

[0049]

[0050] The product solution of Example-1A was cooled with an-ice-waterbath. A sample of chloroacetone (101.8 g, 1.1 mol) was added to thevigorously stirred reaction mixture over about 50 min at between 8 and11° C. After the addition of chloroacetone was completed, the coolingbath was removed and the resulting reaction mixture was allowed to stirat room temperature overnight. The toluene layer was separated, washedwith water (250 mL) and concentrated on a rotary evaporator at 85° C.under house vacuum followed by high vacuum to give the crude titledcompound (225.7 g, 96.7%). ¹H NMR (CDCl₃, 400 MHz) δ 4.95 (bs, 1H), 3.20(m, 4H), 2.54 (t, 2H), 2.20 (s, 3H), 1.35 (s, 9H).

Example-1C[2-[[(4-Methyl-2,5-dioxo-4-imidazolidinyl)methyl]thio]ethyl]carbamicacid, 1,1-dimethylethyl ester

[0051]

[0052] To a 3L 4-neck RB flask equipped with an overhead stirrer, athermocouple and a condenser connected to an empty flask and a caustictrap, was added the product of Example-1B (70 g, 0.3 mol), absoluteethanol (80 mL), sodium cyanide (19.1 g, 0.39 mol), ammonium carbonate(43.3 g, 0.45 mol) and water (720 mL) in this order. The 4^(th) neck wasclosed with a stopper. The resulting reaction mixture was heated atbetween 67 and 68° C. for 6 h. Subsequently, the almost clear brownsolution was cooled to room temperature. Upon cooling, solid began toform and the heterogeneous mixture was stirred at room temperatureovernight. The reaction mixture was then acidified with 12% hydrochloricacid to pH 2 in about 1 h at between −2 and 2° C. The cold reactionmixture was stirred at pH2 for additional 30 min and then filtered. Theflask was rinsed with distilled water (2×250 mL) and each rinse was usedto wash the solid cake. The solid was again washed with distilled water(2×250 mL) and then air-dried for 4 days. The dry solid was trituratedwith 200 mL of toluene for 0.5 h. The slurry was filtered. The solid wasrinsed sequentially with toluene (50 mL) and 1:4 ratio of toluene/hexane(100 mL) and then air-dried at room temperature overnight to give 83.1%yield of the titled compound, m.p. 134-136° C. ¹H NMR (DMSO_(d6), 400MHz) δ 10.62 (s, 1H), 7.85 (s, 1H), 6.83 (m, 0.9H), 6.48 (bs, 0.1H),3.29 (s, 2H), 2.99 (m, 2H), 2.71 (s, 2H), 2.95 (m, 2H), 1.32 (s, 9H),1.24 (s, 3H); ¹³C NMR (DMSO_(d6), 400 MHz), δ 178.1, 157.1, 156.1, 78.4,63.7, 40.7, 39.4, 33.2, 28.9, 23.8.

[0053] Analysis Calcd for C₁₂H₂₁N₃O₄S: C, 47.51; H, 6.98; N, 13.85; S,10.57. Found: C, 47.76; H, 6.88; N, 13.77; S, 10.75.

Example-1D R andS-[2-[[(4-Methyl-2,5-dioxo-4-imidazolidinyl)methyl]thio]ethyl]carbamicacid, 1,1-dimethylethyl ester

[0054]

[0055] The reaction product of Example-IC was separated into its R and Senantiomers on a Chiralpak® AD column eluting with methanol. The Sisomer was the first eluting isomer followed by its R enantiomer. Bothisomers were used in subsequent transformations. S enantiomer:

[0056] [α] in MeOH at 25° C.=+43.0 (365 nm). ¹HNMR: (400 mHz, CD₃OD) δ1.49 (s, 9H), 2.05 (s, 3H), 2.65 (t, 2H), 2.9 (q, 2H, d), 3.20 (m, 2H).IR: λcm⁻¹=1772,1709.

[0057] Analysis calculated for C₁₂H₂₁N₃O₄S (formula weight=303.38): C,47.51; H, 6.98; N, 13.85.

[0058] Found: C, 47.39; H, 6.62; N, 13.83. M+H=304.

[0059] R enantiomer:

[0060] [α]in MeOH at 25° C.=−46.3 (365 nm). ¹HNMR: (400 mHz, CD₃OD) δ1.48 (s, 9H), 2.05 (s, 3H), 2.65 (t, 2H), 2.85 (q, 2H, d), 3.18 (m, 2H).IR: λcm⁻¹=1770,1711.

[0061] Analysis calculated for C₁₂H₂₁N₃O₄S (formula weight=303.38): C,47.51; H, 6.98; N, 13.85.

[0062] Found: C, 48.15; H, 7.04; N, 14.37. M+H=304.

Example-1E S-(2-aminoethyl)-2-methyl-L-cysteine

[0063]

[0064] Acid Hydrolysis Method:

[0065] A 500 mL three-necked round bottom flask equipped with adistillation condenser was charged with the R-isomer product ofExample-ID (45.8 g, 150.9 mmol) and treated portion wise with 48% aq.HBr (160 mL) at room temperature with stirring. After the gas evolutionceased, the mixture was heated with a heating mantle until the pottemperature reached to 126° C. while the volatile t-butyl bromide (bp72-74° C.) followed by a small amount of aq. HBr (approx. 15 mL) weredistilled off. The distillation condenser was replaced with a refluxcondenser and the mixture was heated at reflux for 30 hours. Thesolution was concentrated and the residue was dissolved in water (250mL) and loaded on to a Dowexe 50WX4-200 ion-exchange resin (8.5×11 cm)and eluted with water (2L) followed by dilute aqueous ammonium hydroxide(30 mL of 28-30% ammonium hydroxide diluted to 1000 mL with water, 3L).Fractions containing the desired product were combined, concentrated,and dried under vacuum at 75-80° C. for two hours to give 22.1 g (82%)of the title product, S-(2-aminoethyl)-2-methyl-L-cysteine, as a whitesolid. Proton and C-13 NMR spectra are consistent with the titleproduct. Mp 157° C. ¹H NMR (400 MHz, D₂O) δ 1.19 (3H, s), 2.53 (1H, d,J=13.6 Hz), 2.57-2.72 (2H, m), 2.92 (1H, d, J=13.6 Hz), 2.92 (2H, t,J=6.8 Hz); ³C NMR (100 MHz, D₂O) δ 24.7, 31.3, 38.9, 40.9, 59.6, 180.7.

[0066] Analysis Cald for C₆H₁₄N₂O₂S+0.1H₂O: C, 40.02; H, 7.95; N, 15.56;S, 17.81. Found: C, 39.93; H, 7.98; N, 15.38; S, 17.70.

[0067] Base Hydrolysis Method:

[0068] To a stainless steel autoclave equipped with agitation was added24.2 g (0.08 moles) of the R-isomer product of Example-1D. After purgingthe apparatus with nitrogen, 128 g (0.32 moles) of 10% caustic was addedgenerating a solution. The autoclave was sealed and heated to 120° C.for 30 hours. After cooling to room temperature, the autoclave wasvented to give 142 ml (151 g) of an aqueous solution of the sodium saltof the title product. H¹NMR (sample acidified with HCl and diluted withD₂O, 400 MHz): δ 1.47 (s,3H), 2.75 (m, 2H), 2.90 (d,1H, J=14.8 Hz), 3.06(t, 2H, J=6.4 Hz), 3.14 (d, 1H, J=14.8 Hz). C¹³NMR (sample acidifiedwith HCl and diluted with D₂O, 100 MHz): δ 172.9, 60.8, 39.1, 39.0,30.4, 22.2. MS (MS/CI-LC) M+1 179.

[0069] DBU (218 μL; 1.46 mmol) and ethyl acetimidate hydrochloride (171mg; 1.34 mmol) were dissolved in ethanol (6 mL) in a 25 mL, one-necked,round-bottomed flask at room temperature (˜20° C.). The title product ofExample-1E (200 mg; 1.12 mmol) was added in one portion to thissolution. The mixture was stirred until the title product of Example-1Ewas consumed (1-2 hours). The mixture was chilled with an ice-bath andthen treated with 6 M HCl (830 μL). ¹HNMR analysis indicated a chemicalyield of 95 mole % or better. The solvent was evaporated and the titleproduct of Example-1 was purified by reverse-phase or ion-exchangechromatography.

[0070] A 210 gm solution (containing ˜20 g of the title product ofExample-1E of the base hydrolysis reaction product was put into a 500mL, three-necked, round-bottomed flask. The apparatus was equipped witha mechanical stirrer, a Dean-Stark apparatus (20 mL with a stopcock), acondenser, and a temperature controller. Water (140 mL) was distilledoff from the mixture. 1-butanol (150 mL) was added to the pot and theremaining water (37 mL) was distilled azeotropically. Additional1-butanol (13 mL) was removed by distillation until the pot temperaturereached 117° C. The butanol slurry was cooled to room temperature andfiltered through a pad of celite. The salts were washed with 1-butanol(2×20 mL). DBU (21.8 μL; 146 mmol) and ethyl acetimidate hydrochloride(17.1 mg; 134 mmol) were dissolved in 1-butanol (40 mL) in a 500 mL,three-necked, round-bottomed flask at room temperature. The apparatuswas equipped with a mechanical stirrer, an addition funnel, and atemperature probe. The title product of Example-1E/1-butanol solutionwas put into the addition funnel and added to the ethyl acetimidate/DBUsolution while maintaining the pot temperature below 25° C. The mixturewas stirred until the starting material was consumed (2-3 hours). Asolution of conc. HCl (94 mL) and water (100 mL) was put into a 1 L,three-necked, round-bottomed flask and chilled to 0° C. The apparatuswas equipped with a mechanical stirrer, an addition funnel, and atemperature probe. The reaction mixture was put into the additionfunnel. The reaction mixture was added to the aqueous HCl solution whilemaintaining the temperature below 25° C. Ethyl acetate (100 mL) wasadded to the solution and the layers were separated. The aqueous layerwas washed once more with ethyl acetate (100 mL). ¹HNMR analysisindicated a chemical yield of 95 mole % or better. This title product ofExample-1 was purified by reverse-phase, ion-exchange chromatography,hydrophobic interaction chromatography, or combination thereof. ¹HNMR(400 MHz, D₂O) δ 1.49 (3H, s), 2.08 (3H, s), 2.74 (2H, m), 2.91 (1H, d),3.17 (1H, d), 3.35 (2H, t).

Example 2 Preparation of the Zwitterion

[0071] In an embodiment of the present invention, excess acid may beremoved from the S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteinedihydrochloride concentrate using anion exchange resin. It wasadditionally discovered that the monohydrochloro, free zwitterion, orother fractional acid derivative ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine could be preparedusing the anion exchange resin. The anion exchange method is preferredfor preparing the monohydrochloride and the free zwitterion due to itssimplicity. S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine withless than 0.5 equivalents of acid and low excess salt is especiallyuseful for pharmaceutical preparation of alternative salt forms.

[0072]FIG. 1 shows a schematic representation of the compound titrationcurve. The parent S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteinemolecule has 3 ionizable groups and can exist in 4 ionization states.

[0073] At low pH, the molecule exists as a +2 charged free acid, withthe carboxylic acid, amine and amidine moieties protonated. This is theionization state for the dihydrochloride salt.

[0074] As the pH is increased, the carboxylic acid group is the firstgroup to deprotonate, and this produces a net charge on the molecule of+1. If the pH increase is generated by addition of sodium hydroxide toS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, the sodiumdihydrochloride salt is formed. Other bases would make theircorresponding salt forms. If the increase in pH is due to removal ofchloride ions by anion exchange processing, the product is themonohydrochloride salt with no sodium or other counterions.

[0075] As the pH is further increased, the amine group deprotonates(about pKa=8.4) producing the neutral zwitterionic form of the molecule.A positive charge still resides on the amidine, and a negative chargestill resides on the carboxyl group. In contrast, if such material ismade by the addition of sodium hydroxide to the dihydrochloride, theresulting product is the monohydrochloro sodium salt, mixed with oneequivalent of sodium chloride. The material prepared by the anionexchange resin approach is the free zwitterion.

[0076] Further increases in pH lead to deprotonation of the amidine ion(pKa˜12.5). The molecule in this pH range is the free base and acidsalt. Note that the free base is preferably not prepared by the anionexchange method, since the negatively charged molecule binds with theanion exchange resin.

Example 3 Preparation of Free Zwitterion

[0077] 60 g of Amberlite IRA400 (OH) resin (a strongly anionicquaternary ammonium polystyrene resin, about 16-50 mesh, available fromRohm & Haas, Philadelphia, Pa.,) was prewashed with 4.7 percent (byweight) ammonium hydroxide (50 ml of 28 percent ammonium hydroxide, 250ml deionized water), followed by extensive washing with deionized water.The final conductivity was 6.1 μS.

[0078] Samples containing about 0.9 g ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine di-hydrochloride in142 ml HCl/water solution, were concentrated on a rotary evaporator at60° C. to an oil. To the oil, diluted to 60 ml with deionized water, wasadded aliquots of 0.5 g of washed anion exchange resin while stirring.At five minutes after each aliquot of resin was added, the solution pHwas measured and a sample removed through a syringe filter. A total of 9g of anion exchange resin was added. The final pH was 10.8. The resinwas removed by filtration and the filtrate was concentrated to an oil byrotary evaporation at 60° C.; no solids formed. The starting material,final filtrate and all intermediate samples were assayed by HPLC and ionchromatography for chloride.

[0079]FIG. 2 shows the pseudo-titration curve forS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine in water using theanion exchange resin to adjust pH. The diamond (solid line) is pH andsquare (dashed line) isS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine (percent initial,by ion chromatography). FIG. 3 shows the pseudo-titration curve forS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine in water using theanion exchange resin to adjust pH. The diamond (solid line) is pH andtriangle (broken line) is chloride (by ion chromatography).

[0080] These curves are not true titration curves since samples werewithdrawn during the progress of the reaction, and since trueequilibrium was not attained before the increments of resin were added.Nevertheless, the graphs of FIG. 2 and FIG. 3 illustrate the expectedtrends. As resin is added to theS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine solution the pHrises with change in slope around pH's of 2, 9 and 11. The pH's ofslower rise are representative of the pK's of the carboxylic acid, amineand amidine functional groups, respectively. Above a pH of 10, theS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine concentration insolution decreases. At this point, theS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine is gaining a netnegative charge and is binding to the resin. The chloride results showsome variation between samples but in general show the trend ofdecreasing chloride content with increasing pH. The final chloridecontent is approximately 0.04 mol equivalents. HPLC assay of the samplesshowed no degradation.

Example 4 Removal of Excess HCl to Adjust Acid Equivalents

[0081] To 3.3 g of sample containing around 305 mg/mlS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine dihydrochloride andaround 0.2 eq excess HCl, was added 16.7 g of deioinized water. The pHwas 1.04. To 14 ml of this solution, prewashed Amberlite 400 (OH) resinwas added to obtain a ph of 2.5. The anion exchange treatment lightenedthe color of the solution from light yellow to water white. The resinwas removed by filtration and the starting material and filtrate productwere analyzed by chloride titration and HPLC.

[0082] Qualitative analysis of the starting material and product by HPLCfound no new peaks and no increase in impurities. The results fromchloride analysis by titration show that the chloride was reduced from2.18 equivalents to around 1.14 equivalents. Although not demonstratedhere, the chloride could be adjusted to higher ratio of HCl by additionof HCl.

Example 5 Preparation of Free Zwitterion

[0083] 3.3 g of a sample containing about 1 g ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine di-hydrochloridewas diluted to 20 g. Aliquots of prewashed Amberlite IRA400 (OH) resinwas added to the solution and samples were periodically withdrawnthrough a syringe filter. Intermediate resin filtrations were performedat pH of 7.1 and 8.8 by filtering off the resin in solution and thencontinuing to add fresh resin to the filtrate. This was done to drivethe chloride removal equilibrium and minimize product adsorption. Afterthe final pH of 11.2 was attained, the resin was filtered off. Thestarting materials, intermediate samples and final filtrate wereanalyzed.

[0084] The resulting samples were analyzed by HPLC. No difference wasseen between the HPLC traces of the starting material and product at pHof 11 within a few hours. However, some degradation peaks at around 2-3peak area % were seen in the high pH samples after storage at roomtemperature for around 10 days.

Example 6 Preparation of Free Zwitterion

[0085] Amberlite IRA400 (Cl) resin was rinsed with 3M HCl, water, andthen 3M NaOH. Aliquots were 100 ml per 10 g of resin. This procedure wasrepeated 3 times in order to clean the resin and in order to generatethe OH form. A final rinse with water was carried out until theconductivity of the eluting water was 2 μS. The resin was then used totitrate 40 ml of a 50 mg/ml solution ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine dihydrochloride.The concentration is expressed in terms of zwitterion equivalents.Aliquots were taken throughout the titration, filtered and analyzed byHPLC. Subsamples of the aliquots were saved for a second HPLC analysis 1week after the titration was performed in order to assess the stabilityof the samples. Additional aliquots were taken for Cl analysis using anion selective electrode. The pH was also monitored throughout thetitration.

[0086] The results found in this example mirrored the results found inExample 3. The chloride specific electrode used here to measure the Clcontent produced data that were much less noisy (see FIG. 4). Note thatthe data indicate that in removing 98% of the Cl, a pH of ˜10.85 isreached. More Cl can be removed but this produced significant binding ofcompound to the resin (see FIG. 5). This loss of compound due to resinbinding can be minimized by filtering off the resin toward the end oftitration and replacing a small amount of fresh resin. This practicehelps drive the equilibrium of chloride removal and minimize the sitesavailable for compound loss by binding.

[0087]FIG. 4 Shows titration curves ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine in water withIRA-400 anion exchange resin. FIG. 5 shows the relevant binding dataassociated with increasing pH.

[0088] HPLC analyses were performed using an ion pairing gradientmethod. The method has been shown to detect the presence of thedegradation products that are expected whenS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine is made basic. Ascan be seen in the following table, the data indicate that degradationis not immediate but instead occurs over a period of days. TABLE 2Stability of -[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine freezwitterion. Purity At The Time Of Purity One Week After Sample pHTitration (Peak Area %) Titration (Peak Area %) .94 98.0 98.3 2.13 98.698.4 3.83 98.7 98.1 8.48 98.5 97.4 9.37 98.5 97.2 9.78 98.3 97.2 10.2798.3 96.4 10.83 96.6 94.7 11.6 98.3 92.4 11.75 97.9 89.2

[0089] HPLC Method

[0090] Pump A: 20 mM KH₂PO₄, 10 mM Pentane sulfonic acid, adjusted topH=3 with phosphoric acid

[0091] Pump B: Acetonitrile

[0092] Gradient: 0% B at 0 min, 26% B at 15 min, 0% B at 15.1 min

[0093] Column: YMC ODS-AQ 120 A, 5 μm, 2.6×150 mm

[0094] Wavelength=205 nm

Example 7 Removal of Excess HCl/Preparation of Monohydrochloro2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine.

[0095] In these examples, the chloride removal process was run in batchby stirring the resin, but it could easily be run in a plant setting byrecirculating the S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteinedihydrochloride solution over an anion exchange resin column or an anionexchange membrane. If the pH is inadvertently raised beyond the desiredrange, it may easily be adjusted back by adding an appropriate amount ofHCl. It would be well within the ordinary skill in the art to design alarge scale anion exchange process for this purpose.

[0096] Pharmaceutical Compositions

[0097] Also embraced within this invention is a class of pharmaceuticalcompositions comprisingS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterion inassociation with one or more non-toxic, pharmaceutically-acceptablecarriers and/or diluents and/or adjuvants (collectively referred toherein as “carrier” materials) and, if desired, other activeingredients. The zwitterion form ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine of the presentinvention may be administered by any suitable route, preferably in theform of a pharmaceutical composition adapted to such a route, and in adose effective for the treatment intended. The activeS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterion andcompositions may, for example, be administered orally, intravascularly,intraperitoneally, subcutaneously, intramuscularly or topically.

[0098] For oral administration, the pharmaceutical composition may be inthe form of, for example, a tablet, capsule, suspension or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a particular amount of the active ingredient. Examplesof such dosage units are tablets or capsules. The active ingredient mayalso be administered by injection as a composition wherein, for example,saline, dextrose or water may be used as a suitable carrier.

[0099] The amount of therapeutically active compound that isadministered and the dosage regimen for treating a disease conditionwith the compound and/or compositions of this invention depends on avariety of factors, including the age, weight, sex and medical conditionof the subject, the severity of the disease, the route and frequency ofadministration, and the particular compound employed, and thus may varywidely. The pharmaceutical compositions may contain active ingredientsin the range of about 0.1 to 2000 mg, preferably in the range of about0.5 to 500 mg and most preferably between about 1 and 100 mg. A dailydose of about 0.01 to 100 mg/kg body weight, preferably between about0.5 and about 20 mg/kg body weight and most preferably between about 0.1to 10 mg/kg body weight, may be appropriate. The daily dose can beadministered in one to four doses per day.

[0100] S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterioncan also be administered by a transdermal device. Preferably topicaladministration will be accomplished using a patch either of thereservoir and porous membrane type or of a solid matrix variety. Ineither case, the active agent is delivered continuously from thereservoir or microcapsules through a membrane into the active agentpermeable adhesive, which is in contact with the skin or mucosa of therecipient. If the active agent is absorbed through the skin, acontrolled and predetermined flow of the active agent is administered tothe recipient. In the case of microcapsules, the encapsulating agent mayalso function as the membrane.

[0101] The oily phase of the emulsions of this invention may beconstituted from known ingredients in a known manner. While the phasemay comprise merely an emulsifier, it may comprise a mixture of at leastone emulsifier with a fat or an oil or with both a fat and an oil.Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make-up the so-called emulsifying wax, and the waxtogether with the oil and fat make up the so-called emulsifying ointmentbase which forms the oily dispersed phase of the cream formulations.Emulsifiers and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate,among others.

[0102] The choice of suitable oils or fats for the formulation is basedon achieving the desired cosmetic properties, since the solubility ofthe active compound in most oils likely to be used in pharmaceuticalemulsion formulations is very low. Thus, the cream should preferably bea non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters may be used.These may be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils can be used.

[0103] Formulations suitable for topical administration to the eye alsoinclude eye drops wherein the active ingredients are dissolved orsuspended in suitable carrier, especially an aqueous solvent for theactive ingredients. The active ingredients are preferably present insuch formulations in a concentration of 0-5 to 20%, advantageously 0.5to 10% and particularly about 1.5% w/w.

[0104] For therapeutic purposes,S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterion isordinarily combined with one or more adjuvants appropriate to theindicated route of administration. If administered per os, the compoundmay be admixed with lactose, sucrose, starch powder, cellulose esters ofalkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted orencapsulated for convenient administration. Such capsules or tablets maycontain a controlled-release formulation as may be provided in adispersion of active compound in hydroxypropylmethyl cellulose.Formulations for parenteral administration may be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions and suspensions may be prepared from sterile powders orgranules having one or more of the carriers or diluents mentioned foruse in the formulations for oral administration. TheS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterion may bedissolved in water, polyethylene glycol, propylene glycol, ethanol, cornoil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodiumchloride, and/or various buffers. Other adjuvants and modes ofadministration are well and widely known in the pharmaceutical art.

[0105] The invention being thus described, it is apparent that the samecan be varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the present invention, and allsuch modifications and equivalents as would be obvious to one skilled inthe art are intended to be included within the scope of the followingclaims. For example, a strong anionic resin could be used, or a weakanionic resin could be employed (see Supelco catalog (part ofSigma-Aldridge)-low pressure ion exchange).

What is claimed is:
 1. A method of removing counterions from a saltcompound in a solution, comprising contacting the compound in solutionwith an appropriate ion exchange medium, and separating the solutioncontaining the compound from the ion exchange medium.
 2. The method ofclaim 1 wherein the counterion is a cation, and the ion exchange mediumis an anionic resin.
 3. The method of claim 1 wherein the counterion isan anion, and the ion exchange medium is a cationic resin.
 4. The methodof claim 1 wherein the compound is a synthetic amino acid analog.
 5. Themethod of claim 4 wherein the synthetic amino acid analog includes anamidine functional group.
 6. The method of claim 5 wherein the syntheticamino acid analog is selected from the group consisting ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine, dihydrochloride;S-[2-(ethanimidoylamino)-1-methylethyl]cysteine;(2S,5E)-2-amino-6-fluoro-7-[(1-iminoethyl)amino]-5-heptenoic acid,dihydrochloride; (S,E)-2-amino-2-methyl-6-[(1-iminoethyl)amino]-4-hexenoic acid,dihydrochloride;(2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic acid,dihydrochloride; and(2S,5E)-2-amino-2-methyl-6-fluoro-7-[(1-iminoethyl)amino]-5-heptenoicacid, dihydrochloride.
 7. The method of claim 2 wherein the cationiccounterion is a mineral acid, an organic acid, or a mixture of a mineralacid and an organic acid.
 8. The method of claim 1 wherein the method isperformed with ion exchange resin in a single stirred vessel.
 9. Themethod of claim 1 wherein the method is performed with ion exchangeresin in several batches in a plurality of stirred vessels in series,with intermediate filtering of the resin and replacement with freshresin.
 10. The method of claim 1 wherein the method is performed withion exchange resin where the solution is passed through a resin bedcontained within a column.
 11. The method of claim 1 wherein the methodis performed with an ion exchange membrane. 12.S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterion havingfrom between zero and up to two molar equivalents of a cationiccounterion.
 13. S-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteinezwitterion with between zero and up to 2 molar equivalents ofhydrochloride.
 14. A method of makingS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterioncharacterized by stoichiometrically less than 0.5 equivalents ofhydrochloride ion toS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine comprising:obtaining a source ofS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine dihydrochloride,and removing sufficient hydrochloric acid to obtain theS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine zwitterion havingstoichiometrically less than 0.5 equivalents of hydrochloride ion toS-[2-[(1-Iminoethyl)amino]ethyl]-2-methyl-L-cysteine.
 15. The method ofclaim 14 wherein the hydrochloric acid is removed by a resin in acolumn.